FCF Funded Projects

A gene editing approach to develop new and effective models for FLC

Status: Completed

Timeframe: 2021

Goal: Generate a mouse model of FLC

Principal Investigator: Sean Ronnekleiv-Kelly, MD

Study overview: The purpose of this study was to generate a robust pre-clinical murine model of FLC that can provide a basis for understanding factors contributing to FLC formation, and for therapeutic development. The study proposed to develop a mouse model of FLC using a gene editing approach in a susceptible population, using hydrodynamic delivery of gene editing material to the liver via retro-orbital injection at age 7-8 weeks in mice with varying susceptibility to liver tumor formation. This included C57BL/6 mice, FVB mice, C3H mice, Balb/c mice and DBA/2 mice, which have a range of 2-3 fold lower vulnerability to 3-7 fold higher susceptibility to liver cancer development. In this manner, they wanted to identify if different susceptibilities to liver tumor formation causes earlier onset / more aggressive cancer, which could then ultimately improve the understanding of FLC development. In a second subset of the same mouse strains, they performed retro-orbital injection of the mice at age 2 weeks, a timeframe selected because the fusion gene mutation is an early somatic event in humans (i.e. peak age of FLC diagnosis is 21 years). Most previous studies targeted the DNAJB1-PRKACA mutation to the liver at age 7-8 weeks. The hope was that targeting the gene editing material to generate the fusion gene at 3 – 4 weeks (when liver cells are still actively dividing) would create a different tumor phenotype compared to existing models.

Results: The mouse models created by the introduction of the CRISPR/CAS9 to generate the fusion gene expressed the DNAJ-PRKACA mRNA as well as the fusion protein. Two groups of mice were injected, a control group at 7-8 weeks, which is the typical age of such manipulations and an experimental group at 3-4 weeks, since this age is more representative of the developmental age at which human subjects develop FLC.

The control group was evaluated at age 6 months and investigators did not identify tumor onset macroscopically or microscopically at that time. Some lesions were seen at 6 months of age that were more prominent at 10 months and appeared to match what was reported in a previous publication. The experimental group is being evaluated currently for histopathological abnormalities and tumor development.

Implications: This mouse model will be the first of its kind to express the fusion protein at an earlier time point in development. The development of the fusion protein at an earlier age should ideally give rise to a stronger tumor phenotype.